CN111662939A - Method for promoting burkholderia to synthesize toxoflavin by using signal molecules - Google Patents
Method for promoting burkholderia to synthesize toxoflavin by using signal molecules Download PDFInfo
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- CN111662939A CN111662939A CN202010723120.XA CN202010723120A CN111662939A CN 111662939 A CN111662939 A CN 111662939A CN 202010723120 A CN202010723120 A CN 202010723120A CN 111662939 A CN111662939 A CN 111662939A
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- hdxy
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- SLGRAIAQIAUZAQ-UHFFFAOYSA-N toxoflavin Chemical compound CN1N=CN=C2C1=NC(=O)N(C)C2=O SLGRAIAQIAUZAQ-UHFFFAOYSA-N 0.000 title claims abstract description 242
- 241001453380 Burkholderia Species 0.000 title claims abstract description 70
- 238000000034 method Methods 0.000 title claims abstract description 31
- 230000001737 promoting effect Effects 0.000 title claims abstract description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 52
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 claims abstract description 31
- XEYBHCRIKKKOSS-UHFFFAOYSA-N disodium;azanylidyneoxidanium;iron(2+);pentacyanide Chemical compound [Na+].[Na+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].[O+]#N XEYBHCRIKKKOSS-UHFFFAOYSA-N 0.000 claims abstract description 31
- 239000001103 potassium chloride Substances 0.000 claims abstract description 31
- 229940083618 sodium nitroprusside Drugs 0.000 claims abstract description 31
- 239000011780 sodium chloride Substances 0.000 claims abstract description 26
- 239000001963 growth medium Substances 0.000 claims abstract description 23
- 229910052939 potassium sulfate Inorganic materials 0.000 claims abstract description 18
- 239000001110 calcium chloride Substances 0.000 claims abstract description 17
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 17
- 239000002840 nitric oxide donor Substances 0.000 claims abstract description 13
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 claims abstract description 13
- 239000011734 sodium Substances 0.000 claims abstract description 11
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- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
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- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 4
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- YEESUBCSWGVPCE-UHFFFAOYSA-N azanylidyneoxidanium iron(2+) pentacyanide Chemical compound [Fe++].[C-]#N.[C-]#N.[C-]#N.[C-]#N.[C-]#N.N#[O+] YEESUBCSWGVPCE-UHFFFAOYSA-N 0.000 claims 1
- 238000012262 fermentative production Methods 0.000 claims 1
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- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
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- 108010080698 Peptones Proteins 0.000 description 2
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- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 2
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
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- QDGAVODICPCDMU-UHFFFAOYSA-N 2-amino-3-[3-[bis(2-chloroethyl)amino]phenyl]propanoic acid Chemical compound OC(=O)C(N)CC1=CC=CC(N(CCCl)CCCl)=C1 QDGAVODICPCDMU-UHFFFAOYSA-N 0.000 description 1
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 1
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- 241001225321 Aspergillus fumigatus Species 0.000 description 1
- 241001508395 Burkholderia sp. Species 0.000 description 1
- 229910021592 Copper(II) chloride Inorganic materials 0.000 description 1
- 201000007336 Cryptococcosis Diseases 0.000 description 1
- 241000221204 Cryptococcus neoformans Species 0.000 description 1
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
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- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 1
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
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Abstract
The invention discloses a method for promoting Burkholderia to synthesize toxoflavin by using signal molecules, wherein the signal molecules for promoting Burkholderia to synthesize toxoflavin are Na +, K +, Ca2+ and nitric oxide. The method comprises the following specific steps: performing shake culture on Burkholderia HDXY-02 with the preservation number of CGMCC N0.14054 at 30 ℃ overnight, and inoculating the Burkholderia HDXY-02 into a sterilization culture medium according to the inoculation amount of 1%; then, a certain concentration of signal molecule compounds (NaCl, Na)2SO4、KCl、K2SO4、CaCl2Nitric oxide donor sodium nitroprusside) is added into the culture medium. The invention adds signal components into the culture solutionThe method improves the ability of Burkholderia HDXY-02 in synthesizing toxoflavin and the yield of toxoflavin to 1.32-3.56 times and 1.28-2.21 times of the untreated toxoflavin respectively.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to a method for promoting Burkholderia to synthesize toxoflavin by using signal molecules.
Background
Burkholderia (Burkholderia) is a gram-negative bacterium widely present in water, soil, plant bodies, and human bodies. The Burkholderia can be used for biodegradation, biocontrol, plant growth promotion and the like in agriculture. Part of Burkholderia can synthesize a small molecular antibacterial substance, toxoflavin. Toxoflavin has been shown to have good inhibitory activity against a variety of tumor cells, which are good in antifungal activity, particularly against a mutant strain of Aspergillus fumigatus that is tolerant to azole drugs and cryptococcus neoformans that can cross the blood brain barrier (Xiaoodan Li, Yikui Li, Ren Wang, QizhiWang, Ling Lu. Toxoflavin produced by Burkholeria gladioli from Lycoris a new broad-spectrum fungus. apple Environ Microbiol,2019,85: e 00106-19). A great deal of research shows that some analogues obtained after structural modification of toxoflavin have proved to have good biological activity, and some analogues also show stronger bacterial and fungal inhibitory activity than toxoflavin. Therefore, after the structure of the toxoflavin is modified, the toxoflavin can be used as a candidate drug with good application prospect.
As a small molecular substance having good biological activity, various methods have been tried to synthesize toxoflavin. At present, toxoflavin is mainly produced by adopting a chemical synthesis method, but the chemical synthesis method is technically complex and time-consuming, needs more organic reagents, is easy to pollute the environment, and has low yield so as to cause high production cost. The establishment of a high-efficiency and safe toxoflavin synthesis method is particularly necessary. The biosynthesis has the advantages of environmental protection, low cost and the like. However, the existing biosynthesis method can not be widely applied to the synthesis of toxoflavin. Microorganisms isolated from nature have generally low ability to synthesize toxoflavin, and the yield of 24 hours of fermentation is only about 20mg/L (Levenberg B, Linton SN. on the biosyntheses of toxoflavin, an azapteridine antibacterial produced by Pseudomonas coveenan. J Biol Chem,1966,241: 846-. Researches show that Burkholderia HDXY-02 has the characteristic of high toxoflavin yield, the toxoflavin yield can reach 170mg/L after fermentation for 24 hours in a liquid culture medium, and is much higher than the toxoflavin yield of other strains by 20mg/L, and the obtained product can be used as a production strain for synthesizing toxoflavin by a fermentation method. Further increasing the toxoflavin yield of the strain becomes a problem to be solved by the toxoflavin biosynthesis function. The most suitable fermentation condition and fermentation medium composition for synthesizing the Burkholderia HDXY-02 toxoflavin are determined by a response surface method in the early stage. By optimizing the toxoflavin fermentation method, the yield of toxoflavin can be improved, and the production cost can be reduced.
In addition, microbial secondary metabolite synthesis is a cellular response that is induced by metabolites, nutrients, and other environmental signals, depending on the growth phase, and environmental factors can influence microbial secondary metabolite synthesis. The synthesis of secondary metabolites from different bacteria is influenced by factors such as specific nutrients, temperature, pH, etc.
The transcriptome sequencing result and the fluorescent quantitative PCR result suggest that the difference expression of the signal transduction related genes possibly causes the obvious difference of the Burkholderia HDXY-02 in the yield of the toxoflavin and other strains, and a new idea of promoting the synthesis of the toxoflavin and improving the yield of the toxoflavin by adding signal molecules is provided.
The invention aims to find out a signal molecule capable of obviously improving the synthesis of the berkoehrlichia HDXY-02 toxoflavin and provides a basis for further improving the output of the toxoflavin of the berkohlichia HDXY-02.
Disclosure of Invention
Burkholderia (Latin classification named Burkholderia sp.) HDXY-02 is preserved in China general microbiological Culture Collection Center (CGMCC), the preservation unit address is No. 3 of West Luo No.1 of Beijing Kogyo sunward area, the preservation date is 2017, 4 months and 20 days, and the registration number of the strain is CGMCC No. 14054.
In order to solve the technical problems, the invention adopts the following technical scheme:
adding signal molecule (Na) to culture medium for changing culture medium+、K+、Ca2+And nitric oxide) concentration of compounds (NaCl, Na)2SO4、KCl、K2SO4、CaCl2And nitric oxide donor sodium nitroprusside) to improve the synthesis of toxoflavin and the yield of toxoflavin of Burkholderia HDXY-02 with the number of CGMCC No. 14054.
NaCl and Na were added to the culture medium2SO4、KCl、K2SO4、CaCl2And nitric oxide donor sodium nitroprusside, and analyzing the influence of the addition of the signal molecule compound on the synthesis capability of the Burkholderia HDXY-02 toxoflavin.
The signal molecule compound for improving the synthesis capacity of the Burkholderia HDXY-02 toxoflavin comprises NaCl and Na2SO4、KCl、K2SO4、CaCl2And nitric oxide donor sodium nitroprusside.
The signal molecule compound for improving the synthesis capacity of the Burkholderia HDXY-02 toxoflavin comprises NaCl and Na2SO4、KCl、K2SO4、CaCl2And nitric oxide donor sodium nitroprusside.
The final concentration of the signal molecule compound for improving the synthesis capacity of the Burkholderia HDXY-02 toxoflavin in the culture medium is NaCl and Na2SO4(85mM-256mM), KCl and K2SO4(67mM～201mM)、CaCl 25 mM-50 mM, sodium nitroprusside (10 mu M-1.25 mM).
The concentration of the signal molecule compound for improving the synthesis ability of the Burkholderia HDXY-02 toxoflavin in the culture medium is preferably NaCl (85mM) and Na2SO4(171mM), KCl and K2SO4(67mM)、CaCl2(50mM), sodium nitroprusside (100. mu.M).
The invention relates to a method for promoting Burkholderia to synthesize toxoflavin by using signal molecules, which comprises the following steps:
(1) inoculating activated Burkholderia HDXY-02 into a fresh LB liquid culture medium, performing shake culture at 30 ℃ overnight (14-18h), and inoculating the overnight culture into a sterilization culture medium according to the inoculation amount of 1%;
(2) adding NaCl and Na with certain concentration2SO4、KCl、K2SO4、CaCl2And one or more than two nitric oxide donors of sodium nitroprusside are added into a culture medium, the Burkholderia HDXY-02 is cultured by fermentation, and the culture medium is shake culture at 30 ℃ for 48h, wherein the culture medium is suitable for the growth of the Burkholderia HDXY-02.
The present inventors have found that, in comparison with the comparative examples, the addition of the signal molecule compounds NaCl, Na to the fermentation medium according to the invention2SO4、KCl、K2SO4、CaCl2And the method of nitric oxide donor sodium nitroprusside can effectively improve the synthesis capacity and the yield of toxoflavin of Burkholderia HDXY-02. The present invention further found that when different signal molecule compounds are added in combination, the toxoflavin synthesizing ability of Burkholderia HDXY-02 is higher than that when a single signal molecule compound is added, and a superposition effect is exhibited. Especially when sodium nitroprusside (50 mu M) and KCl (134mM) are added simultaneously, the toxoflavin synthesis capacity of Burkholderia HDXY-02 can be improved to 3.56 times of that of untreated bacteria, and the maximum content of toxoflavin in the fermentation supernatant can reach 2.21 times of that of untreated bacteria. The method improves the yield and the output of the toxoflavin produced by the Burkholderia HDXY-02, solves the problem of low output of the toxoflavin synthesized by microorganisms, can reduce the unit production cost of the toxoflavin, and improves the feasibility of synthesizing the toxoflavin by adopting a microbial fermentation method.
Drawings
FIG. 1 shows the effect of amino acids and inorganic salts on the ability of Burkholderia HDXY-02 toxoflavin synthesis.
FIG. 2 is a toxoflavin standard curve.
FIG. 3 shows NaCl, KCl and CaCl at different concentrations2Influence on the ability of Burkholderia HDXY-02 toxoflavin to synthesize. (A) NaCl; (B) KCl; (C) CaCl2。
FIG. 4 shows different concentrations of Na2SO4、K2SO4P-BoThe influence of the capacity of Klebsiella HDXY-02 toxoflavin synthesis.
FIG. 5 shows the effect of other osmolytes on the capacity of Burkholderia HDXY-02 toxoflavin synthesis.
FIG. 6 shows the effect of nitric oxide on the capacity of Burkholderia HDXY-02 toxoflavin synthesis. (A) The yield of toxoflavin is obtained after exogenous addition of nitric oxide donor Sodium Nitroprusside (SNP) and KCl or simultaneous addition of SNP and KCl; (B) toxoflavin production following exogenous SNP and NO-specific scavenger (cPTIO). OD393/OD600Represents the relative value (OD) of toxoflavin production per cell393Is the ultraviolet absorption characteristic peak value, OD of toxoflavin600Representative cell concentration).
FIG. 7 the effect of exogenous addition of nitric oxide donor sodium nitroprusside on the intracellular c-di-GMP content of Burkholderia HDXY-02.
FIG. 8 is the analysis of the expression level of genes related to synthesis of Burkholderia HDXY-02 toxoflavin by exogenous addition of nitric oxide donor sodium nitroprusside.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
1. Culture medium:
seed medium (g/L): tryptone 10, yeast powder 5 and NaCl 10.
Shake flask fermentation medium (g/L): peptone 20, K2HPO4·3H2O 1.5，MgSO4·7H2O1.5, 15mL of glycerol.
Optimization of shake flask fermentation medium (g/L): 24.24g/L glucose, 20.45g/L peptone, 0.472g/L phenylalanine, K2HPO4·3H2O 1.5，MgSO4·7H2O 1.5。
2. The experimental method comprises the following steps:
(1) activating strains: the Burkholderia HDXY-02 preserved at-80 ℃ and with the preservation number of CGMCC No.14054 is streaked on an LB solid plate and cultured for 24-36 h at 30 ℃.
(2) Inoculation: the activated Burkholderia HDXY-02 is cultured for 14-16 h overnight and is inoculated into a 250mL triangular flask filled with 50mL of culture medium according to the inoculation amount of 1%.
(3) Liquid fermentation: filtering and sterilizing the accelerant, adding the accelerant into a culture medium according to a certain concentration and different combinations, and performing shake culture at 30 ℃ and 200rpm for 48 hours.
(4) And (3) drawing a toxoflavin standard curve: preparing 500mg/L standard sample from toxoflavin (Sigma) with 80% methanol solution, diluting to 24.5mg/L, 18.25mg/L, 12.5mg/L, 6.25mg/L, 5mg/L, 3.75mg/L, 2.5mg/L and 1.25mg/L respectively according to gradient, and detecting the absorbance value (OD 393 nm) at 393nm393) And carrying out regression analysis on the toxoflavin standard sample of each gradient by using Excel to obtain a standard curve and an equation.
(5) Calculating the yield of toxoflavin: centrifuging culture 1mL at 10000g for 1min, extracting the fermentation supernatant with equal volume of chloroform for three times, collecting organic phase, volatilizing, resuspending with 80% methanol solution, and detecting the absorbance (OD) at 393nm393) And calculating the content of the toxoflavin in the supernatant according to a toxoflavin standard curve, wherein three replicates are arranged in each treatment.
(6) The toxoflavin synthesis capacity determination and calculation method comprises the following steps: calculate OD393/OD600(representing the unit capacity for toxoflavin synthesis), three replicates were set up for each treatment.
(7) RNA extraction: with reference to the specification, the operation steps are as follows: the OD value (OD) of each RNA sample at different wavelengths was determined by a spectrophotometer230、OD260、OD280And OD320) And calculating the concentration of the RNA sample. And diluting each extracted RNA sample to a certain multiple, and ensuring that the OD value is between 0.1 and 0.6 so as to ensure the reliability of data. According to the quantitative result, the volume of the added RNA in the reverse transcription system is determined, and the reverse transcription amount of the RNA is ensured to be 2 mu g. cDNA preparation was performed according to the kit instructions.
(8) Real-time fluorescent quantitative PCR:
a. preparing a PCR reaction system: the reaction system was 15. mu.L, where AceQ qPCR SYBR Green Master Mix 7.5. mu.L, primers were 0.5. mu.L each (10 pmol. mu.L)-1) Template cDNA 2. mu.L, in combination with ddH2O is subjected to constant volume of 15 mu L, and lpxC is selected as an internal reference gene;
TABLE 1 primer sequences for related genes
b. Quantitative PCR reaction conditions: the reaction is carried out for 40 cycles at 95 ℃ for 2min, 95 ℃ for 10s and 60 ℃ for 20 s.
(9) The c-di-GMP extraction and detection method comprises the following specific steps:
a. the extraction method comprises the following steps: bacterial culture adjusted to OD600Washing with precooled PBS for 2 times, resuspending the cells with 100 μ L of precooled PBS, treating at 100 deg.C for 5min, adding ethanol to a final concentration of 65%, shaking for 15s, centrifuging at 16000g at 4 deg.C for 2min, transferring the supernatant to a new centrifuge tube, precipitating, extracting for 2 times by the same method, combining the supernatants, drying, and storing at-80 deg.C;
b. the detection method comprises the following steps: adding 200 μ L of ultrapure water heavy suspension extract before detection, filtering, and detecting; HPLC method for determining c-di-GMP: sample size 20 μ L, using C18Column, detection wavelength 253nm, mobile phase for solution A (10mM ammonium acetate aqueous solution) and solution B (10mM ammonium acetate methanol solution), flow rate of 0.2mL/min, gradient elution. Finally, the intracellular c-di-GMP concentration (pmol/mg protein) was calculated based on the c-di-GMP standard curve and the total protein amount (using the modified Lowry method).
EXAMPLE 1 Effect of amino acids, mineral elements on the Synthesis ability of Burkholderia HDXY-02 toxoflavin
According to the fermentation characteristics and physiological metabolic characteristics of the Burkholderia HDXY-02, amino acids and mineral elements are selected and added into a culture medium, and the influence of the amino acids and the mineral elements on the synthesis capacity of the Burkholderia HDXY-02 toxoflavin is analyzed. Relative value of toxoflavin synthesis (OD) in units of bacterial cells393/OD600) To evaluate the ability of Burkholderia HDXY-02 toxoflavin synthesis.
The amino acids were used at the following concentrations:
amino acids: phenylalanine (0.5g/L), tyrosine (0.5g/L), aspartic acid (0.5 g/L).
The selected inorganic salts are as follows: NaCl, KCl, MgSO4、CaCl2、FeSO4、CdCl2、MnCl2、ZnCl2、CuCl2。
As shown in FIG. 1, phenylalanine, tyrosine and aspartic acid did not significantly improve the toxoflavin synthesis ability of Burkholderia HDXY-02. And NaCl, KCl, CaCl2The toxoflavin synthesis capacity of Burkholderia HDXY-02 can be obviously improved by 1.67 times, 1.64 times and 1.52 times when the toxoflavin is added into a culture medium. While adding CdCl2The synthesis of toxoflavin is severely inhibited, and toxoflavin is hardly detected in the fermentation supernatant. When in the culture medium CaCl2After a concentration of more than 50mM, a white precipitate was formed.
The amount of toxoflavin in the fermentation supernatant was calculated from FIG. 2, and it was found that NaCl (85mM), KCl (134mM), CaCl were added2The toxoflavin content in the fermentation supernatant can be increased by 1.35, 1.32 and 1.76 times (50mM), which shows that Na+、K+And Ca2+Promoting toxoflavin synthesis. Furthermore, as shown in FIG. 3, the accelerating effect of NaCl and KCl on the synthesis of H.berghei HDXY-02 toxoflavin gradually decreased with increasing NaCl and KCl concentrations after reaching the peak, which is probably due to the inhibitory effect of higher salt concentrations on the growth of H.berghei HDXY-02. The promotion of toxoflavin synthesis by NaCl and KCl is effective only at low concentrations.
Example 2Na2SO4And K2SO4Effect on the capacity of Burkholderia HDXY-02 toxoflavin Synthesis
NaCl and KCl can improve the synthesis capacity and the toxoflavin yield of the Burkholderia HDXY-02 toxoflavin, in order to clarify Na+And K+For promoting synthesis of toxoflavin, Na is selected2SO4And K2SO4Added into the culture medium according to a certain concentration. As shown in FIG. 4, Na2SO4(85mM-256mM) and K2SO4(67mM-201mM) As with NaCl and KCl, increases toxoflavin synthesis in Burkholderia HDXY-02, Na2SO4The increase times are 1.50, 1.54 and 1.39 times without addition, K2SO4The fold increases were 1.55, 1.39 and 1.34 when no addition was madeAnd (4) doubling. Can increase the toxoflavin content in the fermentation supernatant by 1.28 to 1.63 times respectively. The above results further demonstrate Na+And K+As a signal molecule to promote toxoflavin synthesis.
Example 3 No enhancement of the ability of other permeants to synthesize Burkholderia HDXY-02 toxoflavin
K+And Na+In addition to being a signal molecule, which can cause osmotic stress, in order to confirm whether the increase in the ability of Burkholderia HDXY-02 toxoflavin synthesis is a result of an osmotic pressure change, mannitol (mannitol), sorbitol (sorbitol) and polyethylene glycol (PEG) were added at different concentrations to the medium.
The permeate was used at the following concentrations: mannitol (100mM, 180mM, 340mM), sorbitol (100mM, 180mM, 340mM), PEG (% w/v: 12, 18, 22), NaCl (100mM, 200mM, 300 mM). The osmolarity is consistent for each of the three concentrations of substance.
From FIG. 5, it can be seen that mannitol, sorbitol and PEG do not significantly improve the synthesis ability of toxoflavin of Burkholderia HDXY-02, but inhibit the synthesis of toxoflavin, indicating that NaCl and KCl do not stimulate the synthesis of toxoflavin of Burkholderia HDXY-02 by osmotic stress.
Example 4 Simultaneous addition of nitric oxide and KCl improves the toxoflavin synthesis of Burkholderia HDXY-02
Nitric Oxide (NO) plays a very critical role as an important gas signal molecule in the physiological metabolic process of microorganisms. The relative value (OD) of riboflavin synthesis in units of cells was also determined by adding NO donor Sodium Nitroprusside (SNP) to the culture medium393/OD600) To evaluate the ability of Burkholderia HDXY-02 toxoflavin synthesis.
Nitric oxide donor Sodium Nitroprusside (SNP) was used at final concentration: 10. mu.M, 50. mu.M, 100. mu.M, 250. mu.M, 1.25 mM; KCl was used at a final concentration of 134 mM.
The experimental results show that after SNP treatment, the relative value (OD) of toxoflavin yield per unit of Burkholderia HDXY-02393/OD600) Significantly improved (fig. 6A). The OD can be further improved by adding KCl393/OD600The value is obtained. The 250 μ M SNP promoted the best, 3.06 times that of untreated, but high concentrations of SNP inhibited bacterial growth, so the subsequent experiments were performed with 100 μ M SNP. Increased OD by addition of NO-specific scavenger (cPTIO)393/OD600The values were restored to a level similar to that of the untreated (FIG. 6B), indicating that it is indeed the NO molecule that promotes the toxoflavin synthesis capacity of Burkholderia HDXY-02. The addition of 100. mu.M SNP increased the toxoflavin synthesis capacity of Burkholderia HDXY-02 to around 2 times that of untreated. Meanwhile, by adding 50 mu M SNP and 134mM KCl, the toxoflavin synthesis capacity of Burkholderia HDXY-02 can be improved to 3.56 times of that of untreated bacteria, and the method has a superposition effect, and the maximum content of toxoflavin in fermentation supernatant can reach 2.21 times of that of untreated bacteria.
The NO molecule affects the intracellular c-di-GMP levels of the bacteria. In order to verify whether the NO molecule can change the intracellular c-di-GMP level of the Burkholderia HDXY-02, the intracellular c-di-GMP content of the untreated bacteria and the cells after SNP treatment is determined, and the result shows that the intracellular c-di-GMP content of the Burkholderia HDXY-02 after SNP treatment is obviously improved (figure 7) and is improved by about 2 times compared with the untreated bacteria.
The NO molecule can improve the synthesis of the toxoflavin of the Burkholderia HDXY-02, and further verify whether the transcription level of the related genes for the synthesis of the toxoflavin is changed through fluorescent quantitative PCR (polymerase chain reaction), and the result proves that the transcription level of the related genes for the synthesis of the toxoflavin is remarkably improved (figure 8), which shows that the NO molecule promotes the synthesis of the toxoflavin of the Burkholderia HDXY-02 by improving the transcription of the related genes for the synthesis of the toxoflavin.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
The invention provides technical support for fermentation production of toxoflavin by Burkholderia HDXY-02, and has important production value in future practical application.
Claims (8)
1. A method for promoting the synthesis of toxoflavin by Burkholderia HDXY-02 by using a signal molecule, which is characterized in that the signal molecule can induce the synthesis of toxoflavin and improve the yield of toxoflavin.
2. The method for promoting the synthesis of toxoflavin from Burkholderia HDXY-02 by using signal molecule as claimed in claim 1, wherein the classification is Burkholderia, (b) and (d) andBurkholderiasp.) is deposited in China General Microbiological culture Collection Center (CGMCC), the deposition unit address is No. 3 of Ministry No.1 of West Lu of North West province in the sunny district of Beijing, the microbial research institute of China academy of sciences, the deposition date is 2017, 4 and 20 days, and the registration number of the strain is CGMCC No. 14054.
3. The method of claim 1 for promoting synthesis of toxoflavin from Burkholderia HDXY-02 using a signal molecule, wherein the signal molecule is Na+、K+、Ca2+And nitric oxide.
4. The method for promoting the synthesis of toxoflavin from Burkholderia HDXY-02 by using a signal molecule as claimed in claim 1, wherein the method comprises the steps of:
1) after the Burkholderia HDXY-02 with the preservation number of CGMCC N0.14054 is cultured overnight, transferring the seed solution into a sterilized culture medium by 1 percent of inoculation amount;
2) a signal molecule Na as claimed in claim 3+（NaCl、Na2SO4）、K+（KCl、K2SO4）、Ca2+（CaCl2) Adding one or more of nitric oxide (nitric oxide donor sodium nitroprusside) into the culture solution, and performing shake culture for 48h or more;
3) extracting toxoflavin in the fermentation supernatant by a liquid-liquid extraction method, and calculating the synthesis capacity of the toxoflavin of the Burkholderia HDXY-02 and the content of the toxoflavin in the fermentation supernatant.
5. The method according to claim 4, wherein NaCl, Na2SO4、KCl、K2SO4、CaCl2Sodium nitroprusside, sterilizing with 0.22 μm filter membrane, adding into the culture medium, and adding NaCl and Na in the culture medium2SO4The final concentration is 85mM-256mM, KCl and K2SO4The final concentration is 67mM-201mM, CaCl2The final concentration is 5mM to 50mM, and the final concentration of sodium nitroprusside is 10 μ M to 1.25 mM.
6. The method of claim 4, wherein the liquid-liquid extraction is performed by: centrifuging the fermentation liquid at 8000 r/min for 10 min to remove thallus, adding equal volume of chloroform, extracting for three times, collecting organic phase, volatilizing, re-suspending with 80% methanol solution, and detecting the absorbance (OD) at 393nm393) And calculating the toxoflavin synthesis capacity of the strain and the content of toxoflavin in the fermentation supernatant.
7. The method of claim 4, wherein NaCl, Na2SO4、KCl、K2SO4、CaCl2The final concentrations of sodium nitroprusside in the culture solution were 85mM, 171mM, 67mM, 50mM, and 100. mu.M, respectively.
8. The use of a method of using a signal molecule to promote synthesis of toxoflavin by Burkholderia as claimed in claim 1 in the fermentative production of toxoflavin products.
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